ELISA is an immunoassay that detects either antigen or antibodies in the specimen, by using enzyme–substrate chromogen system for detection.
Principle of ELISA
ELISA is so named because of its two components:
- Immunosorbent: Here, an absorbing material is used (e.g. polystyrene, polyvinyl) that specifically absorbs the antigen or antibody present in serum
- Enzyme is used to label one of the components of immunoassay (i.e. antigen or antibody).
Substrate-chromogen system: A substrate-chromogen system is added at the final step of ELISA.
- The enzyme reacts with the substrate, which in turn activates the chromogen to produce a color
- The classical example is, horseradish peroxidase used as enzyme which reacts with its substrate (hydrogen peroxide), that in turn activates the chromogen (tetramethyl benzidine) to produce a color
- The color change is detected by spectrophotometry in an ELISA reader. Intensity of the color is directly proportional to the amount of the detection molecule (Ag or Ab) present in test serum.
(Ag-Ab complex)-enzyme + substrate → activates the chromogen → color change → detected by spectrophotometry (ELISA reader, Fig. 1A)
Fig1A and B: A. ELISA reader (Biorad); B. ELISA washer. Source: Biorad Pvt. ltd (with permission).
Procedure of ELISA
ELISA is performed on a microtiter plate containing 96 wells (Fig.2), made up of polystyrene, polyvinyl or polycarbonate material.
- ELISA kits are commercially available; contain all necessary reagents (such as enzyme conjugate, dilution buffer, substrate/chromogen, etc.)
- The procedure involves a series of steps done sequentially. At each step, a reagent is being added, and then incubated, followed by washing of the wells [manually or by an automated ELISA washer (Fig. 1B)].
Fig2. ELISA for HBsAg. Source: Department of Microbiology, Pondicherry Institute of Medical sciences, Puducherry (with permission).
Types of ELISA
There are several types of ELISA, which differ from each other in their principles.
Direct ELISA
It is used for detection of antigen in test serum. Here, the primary antibody (targeted against the serum antigen) is labeled with the enzyme.
- Step 1: Wells of microtiter plate are empty, not precoated with Ag or Ab
- Step 2: Test serum (containing antigen) is added into the wells. Antigen becomes attached to the solid phase by passive adsorption
- Step 3: After washing, the enzyme-labeled primary antibodies (raised in rabbits) are added
- Step 4: After washing, a substrate–chromogen system is added and color is measured.
Well + Ag (test serum) + primary Ab-enzyme + substrate- chromogen → Color change (Fig. 3A)
Fig3. A and B: A. Direct ELISA (for antigen detection); B. Indirect ELISA (for antibody detection).
Indirect ELISA
It is used for detection of antibody or less commonly antigen in serum. It differs from the direct ELISA in that the secondary antibody is labeled with enzyme instead of primary antibody. The secondary antibody is an anti -species antibody, e.g. anti-human Ig (an antibody targeted to Fc region of any human Ig). Indirect ELISA for antibody detection is described below (Fig. 3B).
- Step 1: The solid phase of the wells of microtiter plates are precoated with the Ag
- Step 2: Test serum (containing primary Ab specific to the Ag) is added to the wells. Ab gets attached to the Ag coated on the well
- Step 3: After washing, enzyme-labeled secondary Ab (anti-human immunoglobulin) is added
- Step 4: After washing, a substrate-chromogen system is added and color is developed.
Wells are coated with Ag + primary Ab (test serum) + secondary Ab-enzyme + substrate- chromogen → development of color (Fig.3B)
Sandwich ELISA
It detects the antigen in test serum. It is so named because the antigen gets sandwiched between a capture antibody and a detector antibody (Fig. 4A).
- Step 1: The microtiter well is precoated with the capture antibody (monoclonal Ab raised in rabbit) targeted against the test antigen
- Step 2: The test serum (containing antigen) is added to the wells. Ag gets attached to the capture antibody coated on the well
- Step 3: After washing, an enzyme labeled primary ‘detector antibody’ specific for the antigen is added. The detector antibody can be same as the capture antibody
- Step 4: After washing, a substrate–chromogen system is added and color is developed.
Wells coated with capture Ab + Ag (test serum) + primary Ab enzyme + substrate–chromogen → color (Fig. 4A)
Fig4. A and B: A. sandwich ELISA (for antigen detection); B. IgM antibody capture (MAC) ELISA.
IgM Antibody Capture (MAC) ELISA
This is an enzymatically amplified sandwich-type immunoassay. This format of ELISA is widely used for dengue, Japanese encephalitis and West Nile virus, scrub typhus, leptospirosis, toxoplasmosis, etc. (Fig. 4B).
- It is based on capturing primary IgM Ab (in test serum) on a microtiter plate pre-coated with anti-human-IgM Ab, followed by addition of recombinant antigen (e.g. dengue antigen)
- Subsequently, enzyme labelled secondary antibody specific for the antigen is added, followed by addition of substrate-chromogen system
- The use of avidin-biotin system helps in amplifying the signal generated between enzyme-antibody complex, thus increases the sensitivity of the assay.
Wells coated with capture anti-IgM Ab + IgM Ab (test serum) + recombinant antigen + secondary Ab-biotin +avidin-enzyme + substrate–chromogen → color (Fig. 4B)
Competitive ELISA
Competitive ELISA is so named because, antigen in test serum competes with another antigen of the same type coated on well to bind to the primary antibody.
- Step 1: Primary antibody is first incubated in a solution with a serum sample containing the test antigen
- Step 2: This antigen–antibody mixture is then added to the microtiter well precoated with the same type of antigen
- Step 3: The free antibodies bind to the antigen coated on the well. More the test antigens present in the sample, lesser free antibodies will be available to bind to the antigens coated onto well
- Step 4: After washing (to remove free antibodies and antigens), enzyme-conjugated secondary antibody is added
-Step 5: After washing, a substrate–chromogen system is added and color is developed. Intensity of the color is inversely proportional to the amount of antigen present in the test serum (Fig.5).
Fig5. Competitive ELISA for antigen detection.
The competitive ELISA can also be used for the detection of antibody in serum. More so, different formats of competitive ELISA are available such as direct, indirect and sandwich formats. The example given above is an indirect competitive ELISA format used for antigen detection (Fig.5).
ELISPOT Test
It is modification of ELISA that allows the quantitative detection of cells producing antibodies (plasma cells) or cytokines (e.g. lymphocytes or macrophages). ELISPOT is currently used in IGRA (interferon gamma assay), for diagnosis of latent tuberculosis; where the sensitized T cells capable of producing the IFN-γ are measured.
Advantages of ELISA
ELISA is the method of choice for detection of antigens/ antibodies in serum in modern days, especially in big laboratories as large number of samples can be tested together using the 96 well microtiter plate.
- It is economical, takes 2–3 hours for performing the assay
-ELISA has a high sensitivity; that is why, it is commonly used for performing screening test at blood banks and tertiary care sites
- Its specificity used to be low. But now, with use of more purified recombinant and synthetic antigens, and monoclonal antibodies, ELISA has become more specific.
Disadvantages of ELISA
- In small laboratories having less sample load, ELISA is less preferred than rapid tests as the latter can be performed on individual samples
- It takes more time (2–3 hours) compared to rapid tests which take 10–20 minutes
- It needs expensive equipment such as ELISA washer and reader.
Applications of ELISA
ELISA can be used both for antigen and antibody detection.
- ELISA used for antigen detection: Hepatitis B [hepatitis B surface antigen (HBsAg) and precure antigen (HBeAg)], NS1 antigen for dengue, etc.
-ELISA can also be used for antibody detection against hepatitis B, hepatitis C, HIV, dengue, EBV, HSV, toxoplasmosis, leishmaniasis, etc.
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